Responsive luminescent lathanide complexes

ABSTRACT

The invention provides a compound of formula (I): 
                         
(wherein:
         R 1  is an optionally substituted 2-(1-azathiaxanthone);   each —R 2  is independently of the formula —CH 2 —C(═O)—R 4 , wherein R 4  is an amino acid or a salt thereof, attached to the remainder of R 2  through the nitrogen atom of the amino group; and   R 3  is hydrogen or a C 1-6  alkyl group); or
 
(wherein:
   R 1  is an optionally substituted 2-(1-azaxanthone);   each R 2  is independently an optionally substituted glutaric or succinic acid, or a salt or ester thereof; and   R 3  is hydrogen or a C 1-6  alkyl group).

CROSS REFERENCE TO RELATED APPLICATIONS

This is a division of U.S. application Ser. No. 13/145,849, which is thenational stage application under 35 U.S.C. 371 of internationalapplication PCT/GB2010/000147, filed Jan. 29, 2010, which designated theUnited States, was published under PCT Article 21(2) in the Englishlanguage, and claims the benefit of priority of United Kingdom patentapplication 0901556.1 filed Jan. 30, 2009. U.S. application Ser. No.13/145,849 and PCT/GB2010/000147 are incorporated herein by reference,and priority to U.S. application Ser. No. 13/145,849, PCT/GB2010/000147,and UK patent application 0901556.1 is claimed.

FIELD OF THE INVENTION

This invention provides luminescent lanthanide complexes, methods fortheir efficient sensitisation and their use in assays of bioactivespecies, for example present in biological fluids, e.g. solutions and inthe diagnosis of certain medical conditions associated with abnormalquantities of citrate or lactate.

INTRODUCTION

The unique magnetic and spectroscopic properties of the ground andexcited states of the f block ions afford considerable scope for thedevelopment of new chemical entities that can be used as imaging probes,as components of optoelectronic devices, or as key sensor materials.Particular advantages of f-block ions are their intense, line-like andlong-lived luminescence at a range of wavelengths spanning the visibleand near infrared (NIR) regions, which permits time-gated rejection ofunwanted signals arising from (short-lived) auto-fluorescence frombiomolecules. Lanthanide chemistry accordingly plays a key role in suchdiverse areas as display technology and clinical diagnosis.

There is a need for simple modular synthetic routes that lead to stableemissive systems with tunable photophysical properties and high overallquantum yields (>10% for Eu/Tb in competitive media), that resistphoto-fading and bleaching, and can be excited at longer wavelengths tominimise competitive absorption by endogenous molecules or tissue (atthe very least, they should obviate the use of quartz optics). Moreover,they should, preferably, allow scope for conjugation to biomolecules,and should, preferably, be compatible with other probes to permitmultiplexed imaging. Notwithstanding the burgeoning academic literature(e.g. Verhoeven, Bunzli, Raymond and Sammes (for example B H Bakker etal., Coord. Chem. Rev., 2000, 208, 3; JCG Bunzli & C. Piguet, Chem. Soc.Rev., 2005, 34, 1098; S. Petoud et al., J. Am. Chem. Soc., 2003, 125,13324; and A. Dadabhoy et al., J. Chem. Soc. Perkin Trans 2, 2000, 2359)reporting the chemistry of new emissive lanthanide (III) complexes orprobes, no single molecule meets each of these criteria, and newapproaches are required.

Moreover, organic chromophores have been widely used as sensitisers oflanthanide emission. However, very few of these possess a S₁-T₁ energygap small enough to allow excitation at the longest possible wavelengthswithout detrimental back energy transfer from the excited state of themetal ion to the sensitiser T₁ state. This is a particularly demandingtask for the visibly emitting lanthanides, since their high excitedstate energies restrict the range of possible sensitisers to those withrelatively high triplet state energies. Acridones have been used forthis purpose, but in polar media possess an inefficient inter-systemcrossing step, so that sensitiser fluorescence competes with tripletformation.

In WO2006/120444 (University of Durham) it is reported that a number oflanthanide complexes described therein (incorporating azaxanthone andazathiaxanthone sensitisers) undergo efficient sensitized excitation andcan be used in time-resolved assays of bioactive species, especially insignalling the variation in the local concentration of endogenousspecies. Similar or the same complexes and macrocyclic ligands are alsoreported by D. Parker and J. Yu (Chem. Commun. 2005, 3141-3143); J. Yuand D. Parker (Eur. J. Org. Chem., 2005, 4249-4252); and P. Atkinson etal., Org. Biomol. Chem., 2006, 4, 1707-1722).

Two examples of such endogenous species present in biological fluids,and of particular interest, are the oxy anions citrate and lactate.

The citrate anion exists in all living cells. It is not only animportant intermediate in the tricarboxylic acid cycle, but also a keycomponent of fatty acid, cholesterol and hormone synthesis,photorespiration, the glyoxylate cycle and nitrogen metabolism. Due toits metabolic significance, abnormal citrate levels have been linked tothe characteristics of several diseases. For example, citrateconcentration in urine can reflect renal metabolic imbalance. Decreasedurinary citrate excretion has been shown to be important in thepathogenesis of nephrocalcinosis and nephrolithiasis (E. A. Schell-Feitet al., Pediatric Nephrol., 2006, 21, 1830; V. Cebotaru et al., KidneyInt., 2005, 68, 642. 3.). Recently, citrate has been selected as amarker for the discrimination of prostate cancer as a result of thehighly specialized anatomical function of the prostate gland (L. C.Costello and R. B. Franklin, Mol Cancer. 2006, 5, 17; and L. C. Costelloand R. B. Franklin. Prostate Cancer Prostatic Dis. 2008, Jul. 1.).

The main function of the prostate gland is to secrete and store a clearfluid (pH 7.3) that constitutes about 50% of the volume of the seminalfluid that, along with spermatozoa, constitutes the semen. The prostaticfluid is mainly composed of simple sugars, less than 1%, and proteinincluding the prostate-specific antigen (PSA). The secretion alsocontains various amounts of zinc and sodium citrate, whose concentrationchanges significantly as a function of malignancy of the epithelialtissue that makes up this part of the male reproductive organ. Prostatecancer (PCa) is the most common non-skin related male cancer type in theworld, affecting one in ten men in the UK. It is classified asadenocarcinoma; PCa begins when normal semen-secreting prostate glandcells mutate into cancer cells. These may lead to formation of a tumourand eventually, if not recognised, invade nearby organs. As the tumourcells may develop the ability to travel in the bloodstream and lymphaticsystem causing metastasis, PCa is considered as a malignant disease.

Prostate secretory epithelial cells have the specialised function andcapability of accumulating and secreting extraordinarily high levels ofcitrate. This is achieved by the existence of a zinc-inhibited lowmitochondrial aconitase (m-Ac) activity that minimises the ability ofthe cell to oxidise citrate via the Krebs cycle. Consequently, citratesynthesised by these cells is accumulated and secreted, therebyaccounting for the extremely high (20-200 mM) citrate content of humanprostatic fluid, compared with the low values (0.1-0.4 mM) in typicalmammalian cells. The level of prostate m-Ac enzyme appears to be similarto that associated with other cells, although the levels of m-Acactivity and consequent citrate oxidation are significantly lower inprostate cells.

Prostate epithelial cells normally possess uniquely high cellular andmitochondrial zinc levels. Studies have shown that zinc inhibition ofthe m-Ac activity accounts for the minimised citrate oxidation andconsequently the high citrate level which characterises prostate cells.The diminished ability of neoplastic epithelial cells, mainly in theperipheral zone of the prostate, to accumulate zinc (80% decrease inC_(Zn)) is a consistent factor in their development of malignancy andthe subsequent dramatic decrease in prostatic citrate concentration. Thevariation of citrate levels is striking, and is believed to lead to areduction from 180 mM (healthy patients) to around 10 mM as diseaseprogresses.

Prostate cancer screening is an attempt to detect unsuspected cancers intheir earliest stages. As PCa is a slow-growing cancer, the chances toidentify the disease in early stage are high. A major problem involvedin prostate cancer (PCa) diagnosis is the absence of sensitive,accurate, and preferably non-invasive early diagnostic procedures. Thecurrent diagnostic and screening procedures (e.g. the prostate specificantigen test) are considered to be highly inaccurate. Therefore, thereis a need for a uniform, well-established and non-invasive screeningprocedure. Citrate level tests from prostate or seminal fluid samplesmay overcome the risk and inaccuracy of current screening procedures.

One main drawback of citrate level measurement in the prostate fluid isthat obtaining the sample requires a biopsy. Prostate fluid, as the mainsource (about 50%), gives rise to seminal fluid citrate level of about20-60 mM. The high citrate content of seminal fluid is consistent withits function as a buffer to maintain the pH of semen. It may also serveas a chelator for zinc and other divalent cations which are highlyconcentrated and involved in liquefaction of semen, as well as servingas an energy source for sperm maturation and viability. Its role may becomplex, but importantly the citrate level in seminal fluid isproportionate to that in the prostate fluid. Therefore, citrate levelmeasurements from seminal fluid samples could aid the detection ofprostate malignancy, in particular, analysis of citrate concentration ofexpressed prostatic fluid can provide a simplified, accurate andrelatively non-invasive screening procedure useful in the diagnosis ofprostate cancer. (L. C. Costello and R. B. Franklin. Prostate CancerProstatic Dis. 2008, Jul. 1., infra).

It is also notable that a sensor possessing an appropriate citrateaffinity may be used to analyse urine samples. This may aid in thedetection of any renal anomaly, such as urolithic kidney dysfunction ornephrolithiasis, as the citrate concentration in urine (about 4 mM in ahealthy patient) may be significantly increased.

Lactic acid (2-hydroxypropanoic acid) is a chemical compound that playsa significant role in several biochemical processes. It is a chiralalpha hydroxy acid. The concentration of blood lactate in humans isusually 1-2 mM at rest, but can rise to over 20 mM during intenseexertion. Lactic acid has two enantiomeric forms, L(+) and D(−);L(+)-lactic acid is the naturally occurring isomer and is present in thehuman body. Lactic acid is a weak acid (pK_(a)=3.86) and underphysiological conditions the majority of lactic acid is present aslactate anion.

Lactic acid is naturally present in many foodstuffs and is formed bynatural fermentation in products. It is also used in a wide range offood applications such as bakery products, beverages, meat products,confectionery, dairy products, salads, dressings and ready meals. Lacticacid in food products usually serves as either as a pH regulator,preservative or as a flavouring agent (E270). It also has several otherimportant applications and roles in many aspects of our daily life, suchas pharmaceutical and cleansing products or as a precursor forbiodegradable polymers. In animals and humans, L-lactate is constantlyproduced from pyruvate via the enzyme lactate dehydrogenase (LDH) in aprocess of fermentation during normal metabolism and exercise. It iswell known that it is formed from glycogen by muscle cells when theoxygen supply is inadequate to support energy production. Accumulationof lactic acid in the muscle, occurs only during short bouts of exerciseof relatively high intensity, is often related to fatigue and musclesoreness. Lactic acid levels also increase in conditions such as heartfailure, severe infection (sepsis), or shock. In each case, the flow ofblood and oxygen throughout the body is lowered. Lactic acid levels arealso elevated when the liver is severely damaged or diseased, becausethe liver normally breaks down lactic acid. Very high levels of lacticacid may cause a serious, sometimes life-threatening condition termedlactic acidosis. Due to its key role not only in everyday products butalso in the human body, the development of a single component, fastresponse lactic acid sensor is needed. The assay is of particularimportance for confirmation of the hypothesis (Warburg effect) ofelevated lactic acid levels in malignant (cancerous) tissue samplesand/or biological fluids.

Current commercially available (L-) lactic acid assays are based onenzymatic methods. The quantification of L-lactic acid requires twoenzyme reactions catalysed by L-lactate dehydrogenase (L-LDH), whereL-lactate is oxidised to pyruvate by nicotinamide-adenine dinucleotide(NAD⁺), (Eq.1)

$\begin{matrix}{{L\text{-}{Lactate}} + {N\; A\;{D^{+}\overset{L\text{-}L\; D\; H}{\longrightarrow}{pyruvate}}} + {N\; A\; D\; H} + H^{+}} & \left( {{Eq}{.1}} \right)\end{matrix}$

However, since the equilibrium of Eq.1 lies firmly in favour of L-lacticacid and NAD⁺, a further reaction is required to ‘trap’ the pyruvateproduct. This is achieved by the conversion of pyruvate to D-alanine and2-oxoglutarate, using the enzyme D-glutamate-pyruvate transaminase(D-GPT) in the presence of a large excess of D-glutamate, (Eq.2).

$\begin{matrix}{{Pyruvate} + {D\text{-}{{glutamate}\overset{D\text{-}G\; P\; T}{\longrightarrow}D}\text{-}{alanine}} + {2\text{-}{oxoglutarate}}} & \left( {{Eq}{.2}} \right)\end{matrix}$

The amount of NADH formed in the above coupled reaction isstoichiometric with respect to L-lactic acid. It is the NADH which ismeasured by an increase in absorbance at 340 nm. The assay is specificto L-lactic acid and linear over the range of 0 to 3.3 mM; moreconcentrated samples requires dilution prior to analysis. The smallestdifferentiation in absorbance for the assay is 0.01 absorbance units.This corresponds to a 2.4 micromolar difference in L-lactic acidconcentration in the solution (with a sample volume of 1.5 mL).

This method of analysis has been found to be sensitive to certaininterferents. Analysis of acidic samples should be performed afterraising sample pH to 10 (using NaOH), following incubation for 30 min.Samples containing carbon dioxide, need to be degassed by increasing topH to 10 with constant stirring. Coloured samples may also causeinterference. Strongly coloured samples require treatment by acombination of surface adsorbents and filtration. Samples containing fatrequires elimination of any solid components and clarification. Samplescontaining protein require elimination (digestion and filtration orcentrifugation) of any high molecular weight material. Thus, whilst themethod is accurate, it is based on a relatively insensitive measurementof absorbance at 340 nm. The need to treat certain samples prior toanalysis adds complexity. As with any UV absorbance-based method, areasonable amount of sample is required to execute a measurement. Thisfeature combined with the observed insensitivity below L-lactic acid atlevels of 0.5 mM prevents the assessment of any biological fluid with asmall sample volume or a low lactic acid concentration. Being afour-component enzymatic assay kit, it also requires time and care toset up, and the kit requires cold storage (−20° C.) and rather long dataacquisition times (sometimes up to 30 min reaction time involving 3-4readings).

There remains a need to develop simple chemoselective methods todetermine the concentration of these oxyanions—i.e. citrate andlactate—in biological fluids such as serum, urine and prostate orseminal fluid samples.

Considering the analysis of citrate and lactate in bio-fluids, asingle-component, fast response sensor for each species is desirable.This should ideally possess the following characteristics: tuneableaffinity for the target analyte; insensitivity to common interferencesfound in typical bio-fluid samples such as anions, proteins and certaincations (e.g. Ca²⁺, Zn²⁺ and Mg²⁺) that perturb the equilibriumspeciation; allow multiple readings using emission spectral techniques,preferably in the visible or near-visible range of the spectrum; andcapability to operate on small sample volumes.

SUMMARY OF THE INVENTION

We have surprisingly found that lanthanide complexes of certainmacrocycles are of particular utility in allowing selective detection ofthe citrate and lactate anions in a variety of liquid media. Inparticular, the present invention arises from the surprising findingthat certain lanthanide complexes of azaxanthone- orazathiaxanone-derivatised macrocyclic ligands show specificity withregard to the reversible binding of citrate and lactate anions. We havethus found that a solution to the problem of measuring the concentrationof certain citrate and lactate in biological fluids may be achieved withcertain emissive lanthanide complexes, based on functionalisedmacrocyclic ligands, as defined herein, that are able to bind to citrateand lactate selectively and to signal this binding event by modulationof the metal-based emission, allowing (typically) ratiometric changes inband intensity to be monitored as a function of anion concentration. Theaffinity and selectivity profile of a given complex cannot be predictedprecisely a priori, on account of the complex interplay between theeffects of charge, steric demand and competitive ligation and thesystems defined herein possess profiles that exhibit unexpectedly highselectivity for lactate or citrate respectively.

Viewed from a first aspect therefore the invention provides a compoundof formula (I):

(wherein:

R¹ is an optionally substituted 2-(1-azathiaxanthone);

each —R² is independently of the formula —CH₂—C(═O)—R⁴, wherein R⁴ is anamino acid or a salt thereof, attached to the remainder of R² throughthe nitrogen atom of the amino group; and

R³ is hydrogen or a C₁₋₆ alkyl group); or

(wherein:

R¹ is an optionally substituted 2-(1-azaxanthone);

each R² is independently an optionally substituted glutaric or succinicacid, or a salt or ester thereof; and

R³ is hydrogen or a C₁₋₆ alkyl group).

Viewed from a second aspect the invention provides a complex comprisinga compound of formula (I) and a lanthanide ion, in particular alanthanide (III) ion.

Viewed from a third aspect the invention provides the use of a complexof the invention, wherein R¹ is an optionally substituted2-(1-azathiaxanthone); and each —R² is independently of the formula—CH₂—C(═O)—R⁴, in the analysis of citrate present in a sample ofinterest. Complexes of the present invention of use in this aspect ofthe invention are referred to herein as comprising a compound of formula(Ia).

Viewed from a fourth aspect the invention provides the use of a complexof the invention, wherein R¹ is an optionally substituted2-(1-azaxanthone); and each R² is independently an optionallysubstituted glutaric or succinic acid, or a salt or ester thereof, inthe analysis of lactate present in a sample of interest. Complexes ofthe present invention of use in this aspect of the invention arereferred to herein as comprising a compound of formula (Ib).

Viewed from a fifth aspect the invention provides a method of analysingcitrate present in a sample of interest, the method comprising:

-   -   (i) contacting the sample of interest with a complex of the        invention comprising a compound of formula (Ia);    -   (ii) exciting the azathiaxanthone; and    -   (iii) determining the quantity or concentration of any citrate        in the sample of interest by analysis of the modulation in one        or more emission bands resultant from the exciting where citrate        is present.

Viewed from a sixth aspect the invention provides a method of analysinglactate present in a sample of interest, the method comprising:

-   -   (i) contacting the sample of interest with a complex of the        invention comprising a compound of formula (Ib);    -   (ii) exciting the azaxanthone; and    -   (iii) determining the quantity or concentration of any lactate        in the sample of interest by analysis of the modulation in one        or more emission bands resultant from the exciting where citrate        is present.

Viewed from a seventh aspect, the invention provides a method of, or foruse in, the screening or diagnosis of prostate cancer comprising:

-   -   (i) obtaining a liquid sample from a subject;    -   (ii) optionally diluting the liquid sample; and    -   (iii) practising a method according to the fifth aspect of this        invention, wherein the liquid sample or the diluted liquid        sample constitutes the sample of interest.

Viewed from a eighth aspect, the invention provides a method of, or foruse in, the screening or diagnosis of a renal anomaly comprising:

-   -   (i) obtaining a liquid sample from a subject;    -   (ii) optionally diluting the liquid sample; and    -   (iii) practising a method according to the fifth aspect of this        invention, wherein the liquid sample or the diluted liquid        sample constitutes the sample of interest.

Viewed from a ninth aspect, the invention provides a method of, or foruse in, the screening or diagnosis of excessive lactic acid comprising:

-   -   (i) obtaining a liquid sample from a subject;    -   (ii) optionally diluting the liquid sample; and    -   (iii) practising a method according to the sixth aspect of this        invention, wherein the liquid sample or the diluted liquid        sample constitutes the sample of interest.

Other aspects and embodiments of the invention will be apparent from thediscussion and non-limiting exemplification of the invention thatfollows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1I show variations representative LC chromatogramsfollowing HPLC analysis/purification of complexes of the invention andcomparative complexes.

FIG. 2. shows a 50 μs time-gated high resolution Eu emission spectrumobtained by exciting mixtures of varying concentrations of sodiumcitrate with a europium-containing complex (20 μM) of the invention([EuL³]⁺). The other conditions are as described for FIG. 3. below. Theinsert shows the ratio of two emission bands as a function of citrateconcentration, with the fit (line) to the data.

FIG. 3 shows high resolution Eu³⁺ emission bands obtained by excitingmixtures of varying concentrations of sodium lactate with aeuropium-containing complex of the invention ([EuL⁹]⁻) (pH 6.5, 0.1 MHEPES, λ_(exec) 337 nm, 10 μM complex, 0.1 M NaCl, 0.3 mM NaHCO₃, 0.4 mMKCl, 0.03 mM HSA, 0.4 mM CaCl₂, 0.2 mM ZnCl₂, 0.5 mM MgCl₂). The insertshows the ratio of two emission bands as a function of lactateconcentration, with the fit (line) to the data.

FIG. 4 shows high resolution Eu emission spectra, of the europiumcomplex [Eu.L³]⁺ (pH 6.5, 0.1 M HEPES, 298K, λ_(ex)=380 nm, C_(EuL)=10μM) as a function of sodium citrate concentration, displaying (insertleft) the calibration curve determined using the given emission bandintensity ratios (614 vs. 683 nm) as a function of sodium citrate.(insert right) time gated (10 μs) Eu emission spectra, of the europiumcomplex (pH 6.5, 0.1 M HEPES, 298K, 2_(ex)=365 nm, C_(EuL)=10 μM) as afunction of sodium citrate concentration.

FIG. 5 shows a comparison of citrate concentrations determined indiluted fluid samples, in particular prostate fluid samples, comparingthe results obtained according to the present invention with those usinga citrate lyase kit, (R²=0.9939).

FIG. 6 shows a comparison of lactate concentrations determined indiluted fluid samples, in particular prostate fluid samples, comparingthe results obtained according to the present invention with those usinga L-Lactate dehydrogenase kit, (R²=0.9956).

FIG. 7 shows some structures of europium (III) complexes of theinvention and comparative complexes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention exploits the phenomenon of long-lived emissionfrom lanthanide ions after excitation, luminescence that persists forseveral milliseconds and, in particular, the modulation of the lifetimeand spectral form of the luminescence resultant from reversibledisplacement of co-ordinated water molecules or other more weakly boundmolecules or anions. The anion binds reversibly in aqueous media to thelanthanide centre, altering the local coordination environment, leadingto changes in emission spectral form, for example altering the relativeintensity of several emission bands (see e.g. R. S. Dickins et al, J.Am. Chem Soc., 2002, 124, 12697). By using time-gated spectralacquisition methods known to those of skill in the art, autofluorescencefrom samples of interest, luminescence from biomolecules found in suchsamples and undesired ligand fluorescence can be eliminated.

The compounds of formula (Ia) comprising azathiaxanthone moieties havebeen found to exhibit unexpectedly high selectivity for the citrate ionmaking use of chelation of the hydroxyl and α-carboxylate group by wayof reversible displacement of coordinated water molecules. The compoundsof formula (Ib) comprising azaxanthone moieties have been found to beselective towards binding of the lactate ion.

In the compounds of formula (Ia), R¹ is an optionally substituted2-(1-azathiaxanthone) molecule, that is to say a 1-azaxanthone moleculeattached to the remainder of the compound at its 2-position. In thecompounds of formula (Ib), R¹ is an optionally substituted2-(1-azaxanthone) molecule. Where substituted, substituents may bepresent, for example, at one or more of the 3, 7, 8 or 9 positions ofthe 1-aza(thia)xanthone moiety.

One or more substituents may be present on the two aromatic rings of theaza(thia)xanthone and these substituents may be, for example,independently selected from alkyl, acylamino, amido, carboxylate andester substituents.

By alkyl is meant herein a saturated hydrocarbyl moiety that maycomprise straight-chain, branched or cyclic regions. Typically an alkylwill comprise from 1 to 20 carbon atoms, more typically from 1 to 6carbon atoms.

By aralkyl is meant herein an aryl or heteroaryl-substituted alkylmoiety. Typically the (hetero)aromatic moieties in aralkyl substituentsare monocyclic, for example phenyl or monocyclic heteroaryl moietiessuch as pyridyl, furanyl, thiophenyl etc. Aryl or heteroaryl moietiesmay be substituted e.g. with one or more alkyl, alkyloxy (i.e.alkyl-O—), halo, nitro, amino, carboxy and ester substituents.

By acylamino is meant a substituent of formula —NHCOR, wherein R is thiscontext is alkyl, aryl or heteroaryl and the hydrogen atom indicated isoptionally replaced, but typically is not, with an alkyl, aryl orheteroaryl moiety. By amido is meant a substituent of formula —C(O)NH₂in which one or both, typically one, of the hydrogen atoms indicated maybe replaced with alkyl, aryl or heteraryl.

By carboxyl is meant a substituent of formula —CO₂H. A carboxylsubstituent may, if desired, be derivatised to form esters of formulaCO₂R, wherein R is this context is alkyl or aryl or heteroaryl, or amidosubstituents.

Often the 2-(1-aza(thia)xanthone) moiety will be unsubstituted or, ifsubstituted, is substituent once or twice, e.g. once, with an alkyl,acylamino or carboxy, ester or amido substituent. A detailed descriptionof how to access such derivatised 1-aza(thia)xanthone molecules isdescribed by P. Atkinson et al. (Org. Biomol. Chem., 2006, 4 1707-1722).

The R⁴ substituents in compounds of formula (Ia) may be selectedindependently, but are typically the same, and comprise an amino acid orsalt thereof, attached to the remainder of R² through the nitrogen atomof the amino group.

By amino acid is meant herein a molecule that comprises both an aminoand a carboxylic acid functionality. Typically the amino acids employedaccording to this invention are, but need not be, naturally occurringamino acids comprising amino acid and carboxylic acids attached to acommon carbon atom (the so-called α-carbon atom). As is known aminoacids (natural or otherwise) may comprise additional amino or carboxylicacid moieties because of which the amino and carboxylic acidfunctionalities attached to the α-carbon atom are referred to as theα-amino and α-carboxylic acid groups. Typically R⁴ is an amino acid, orsalt thereof, (in particular a carboxylate salt, in which the carboxylicacid of the carboxyl group is deprotonated) attached to the remainder ofR² through the nitrogen atom of the α-amino group.

The R⁴ moieties, typically, are conveniently obtained from naturallyoccurring amino acids, for example phenylalanine or alanine (wherein —R⁴moieties are —N(H)C(H)(CH₂Ph)COOH (or a salt thereof) and—N(H)C(H)(CH₃)COOH (or a salt thereof) respectively). Other amino acids(including naturally occurring amino acids) are known to those skilledin the art. Examples of naturally occurring amino acids include alanine,arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, serine, threonine, tryptophan, tyrosine and valine.

As a consequence of R⁴ being attached to the remainder of R² through anamino group (typically via the a-nitrogen atom) the (or a) carboxylicacid of the amino acid group R⁴ remains unreacted. As a consequence, thecarboxylic acid is susceptible to derivatisation. Accordingly, thecompounds of this invention of formula (Ia) may comprise the carboxylicacid of the amino acid as the free acid, or as the salt of the freeacid, for example as a sodium, potassium or other convenient salt. Inthe complexes of the invention, the lanthanide ion may serve as thecounterpart cation to the anionic carboxylate moieties, where thecarboxylic acids of R⁴ are deprotonated (as they are when present insalts of the free acids).

A particularly surprising feature of the present invention in relationto the compounds of formula (Ia), and complexes comprising thesecompounds, is the elevation of the apparent stability of ternarycomplexes of cationic lanthanide-containing complexes with citratewherein the carboxylic acid moiety present in the R⁴ substituents isesterified, (whereby to provide compounds not of the invention) in thepresence of certain concentrations of divalent metals such as magnesium,zinc and calcium. Surprisingly, it has been found that carboxylatecomplexes in which the carboxylic moiety is not esterified (complexes ofthe invention) constitute complexes in which the stabilities of theternary complexes are not elevated to such an extent. Furthermore, useof unesterified complexes gives rise to affinity and selectivityprofiles that are particularly useful for the analysis of typicalconcentrations of citrate (for example over the range of 0.1 to 2 mM,after sample dilution by ×100) found in typical samples of interest(such as clinical samples). Notably, complexes analogous to those of thepresent invention in which the carboxylic acid groups of the R⁴ moietiesare esterified have been surprisingly found to possess too high abinding affinity to allow modulation of the emission spectral responseover the target working range of citrate concentrations found in typicalcitrate-containing samples of interest. The foregoing is manifested inthe data presented below (see Table 1), where the apparent affinityconstant of a comparative example of the invention, comprisingesterified R⁴ substituents, increases by about two log units is thepresence of 2 mM MCl₂ (M=Ca, Zn and Mg).

Finally, in the compounds of formula (Ia), substituent R³ may be eitherhydrogen or a C₁₋₆ alkyl group. Where the R³ substituent is other thanhydrogen, the alkyl group may be, for example, a straight-chain orbranched alkyl group, and in particular a straight-chain or branchedC₁₋₄ straight chain or branched alkyl group such as a methyl, ethyl,n-propyl or iso-propyl or tert-butyl group. In certain embodiments ofthe invention R³ in compounds of formula (Ia) is a methyl group. Suchalkylated compounds may be prepared by Eschweiler-Clarke reaction of theparent secondary amine, exemplification of which is providedhereinafter. Typically, however, R³ is hydrogen in compounds of formula(Ia) or complexes derived therefrom.

Examples of compounds of the invention of formula (Ia) in which thecompounds are incorporated into lanthanide (III)- (e.g. Eu (III)-)containing complexes are depicted in FIG. 7 (ligands L³ and L⁴).Examples of compounds of the invention of formula (Ia) as thus of eitherof the following formulae:

(wherein each -A is —CO₂H, or a salt thereof).

In the compounds of formula (Ib), R¹ is an optionally substituted2-(1-azaxanthone) molecule as already described in detail hereinabove.

In compounds of formula (Ib), each R² is independently an optionallysubstituted glutaric or succinic acid, or a salt or ester thereof.Typically, both R² substituents will be the same. As is known by thosewith knowledge of the art, glutaric acid is a 5-carbon atom-containingdicarboxylic acid with the disposition of the carboxylic acids being ateither end of a linear, saturated and unsubstituted hydrocarbyl chain.Succinic acid is an analogue of glutaric acid containing one lessmethylene group in the linear saturated, hydrocarbyl chain. The positionat which the glutaric or succinic acid is connected to the nitrogen atomin the compounds of formula (Ib) is typically at a carbon atom to whicha carboxylic acid is connected, i.e. as a so-called α-carbon atom. Withsuccinic acid, therefore, this typical point of connectivity is ateither of the two carbon atoms to which the terminal carboxylic acidsare connected; in glutaric acid, the point of connectivity is thustypically at a carbon atom other than the central carbon atom of thethree carbon atom-containing chain linking the terminal carboxylicfunctionalities. The glutaric or succinic acids that may be employed inthe compounds of formula (Ia) are typically unsubstituted, although adegree of substitution may be tolerated, with, for example, one or morehalo, nitro, amino, hydroxyl or C₁₋₆ alkyl or C₂₋₆ alkenyl or alkynylsubstituents present.

Typically, the compounds of the invention of formula (Ib) compriseglutaric acid (i.e. unsubstituted glutaric acid) as each R², or a saltor ester thereof. Examples of compounds of the invention of formula (Ib)in which the compounds are incorporated into lanthanide (III)- (e.g. Eu(III)-) containing complexes are depicted in FIG. 7 (ligands and L⁸ andL⁹). Examples of compounds of the invention are thus of the followingformula:

(wherein each -A is —CO₂H, or a salt thereof; and R³ is either hydrogenor methyl). As will be understood by those skilled in the art, thecarboxylic acid functionalities present in the glutaric or succinicacids of moieties R² may be present as the free acids, as salts, forexample sodium or potassium salts, or esters, for example C₁₋₆ alkylesters such as methyl or ethyl esters. Advantageously, where themacrocyclic compounds of formula (Ib) are part of alanthanide-containing macrocyclic complex, the carboxylic acidfunctionalities are present in deprotonated form (i.e. as salts). Inthis way, each dicarboxylic acid substituent R² gives rise to thecoordination to the lanthanide ion of the a-carboxylate (i.e. thecarboxylate attached to the carbon atom attached to the nitrogen atompresent in the compounds of formula (Ib) to which the substituents R²are connected) and one non a-carboxylic acid. In addition, one of theother carboxylic groups in one of the R² ligands may bind to thelanthanide ion in the complex. Competitive displacement of thisnon-a-carboxylate by binding with lactate in particular can occur andchanges the coordination environment the lanthanide centre, this changeto the coordination environment giving rise to the detectable modulationin emissivity of radiation upon excitation of the resultant lanthanidecomplex.

Finally, in the compounds of formula (Ib), substituent R³ may be eitherhydrogen or a C₁₋₆ alkyl group. Where the R³ substituent is other thanhydrogen, as is often advantageous (but not necessary) with compounds offormula (Ib), the alkyl group may be, for example, a straight-chain orbranched alkyl group, and in particular a straight-chain or branchedC₁₋₄ straight chain or branched alkyl group such as a methyl, ethyl,n-propyl or iso-propyl or tert-butyl group. In certain embodiments ofthe invention R³ in compounds of formula (Ib) is a methyl group. Suchalkylated compounds may be prepared by Eschweiler-Clarke reaction of theparent secondary amine, exemplification of which is providedhereinafter. Advantageously, lanthanide-containing complexes comprisingcompounds of formula (Ib), wherein R³ is a C₁₋₆ alkyl group, such asthose described immediately hereinbefore, are less hydrated in solutionand have increased steric demand at the lanthanide ion.

It will be understood by those skilled in the art that some of thecompounds and complexes of the invention can exist in the form of one ormore stereoisomers. Individual of mixtures of stereoisomers are embracedby the references herein to the compounds and complexes of theinvention.

Exemplary compounds of the invention, which are described in theexamples which follow below are1,7-bis(α-dimethylglutarate)-4-[(1-azaxanthone)-2-methyl]-1,4,7,10-tetraaza-cyclododecane,1,7-bis(α-glutarate)-4-[(1-azaxanthone)-2-methyl]-1,4,7,10-tetraaza-cyclododecane,1,7-bis(α-dimethylglutarate)-4-[(1-azaxanthone)-2-methyl]-10-methyl-1,4,7,10-tetraaza-cyclododecane,1,7-bis(α-glutarate)-4-[(1-azaxanthone)-2-methyl]-10-methyl-1,4,7,10-tetraaza-cyclododecane,(SS)-1,7-bis(carboxy-2-ethylcarbamoylmethyl)-4-[(1-azathiaxanthone)-2-methyl]-1,4,7,10-tetraazacyclododecaneand(SS)-1,7-Bis(carboxy-2-ethylcarbamoylphenyl)-4-[(1-azathiaxanthone)-2-methyl]-1,4,7,10-tetraazacyclododecane.

As described above, the compounds of formula (I) of this invention maybe used to provide luminescent lanthanide complexes by binding to alanthanide ion, in particular a lanthanide (III) ion. The lanthanide ionmay be selected from the following: Ln(III), Ln=Nd, Sm, Eu, Gd, Tb, Dy,Ho, Er, Tm, Yb and is typically Eu(III) or (Tb(III), in particularlyEu(III). In the complexes of the invention in which the compounds offormula (I) are complexed with a lanthanide ion, the direct coordinationof the chromophore (i.e. either an azathiaxanthone or azaxanthone) tothe central lanthanide ion minimises the separation between theazathiaxanthone or azaxanthone sensitiser and the acceptor lanthanideion ensuring efficient energy transfer and efficient luminescence.

The resultant complexes are particularly effective in the selectiveanalysis, i.e. detection and/or quantification, in samples of interest,typically obtained from living beings, in particular a mammal, mostparticularly a human, in order to analyse for citrate or lactate. Suchsamples may be of blood, plasma, serum, urine, saliva, mucus,perspiration, lymph, gastric juice, aqueous humour, semen or prostaticfluid. Such samples may be amniotic, pericardial, peritoneal, pleural,cerebrospinal, vaginal or faecal. Typically samples are of serum, urine,saliva, seminal or prostate fluid samples. Where such samples, typicallyliquid samples, are obtained from a human or an animal body, it will beappreciated that the actual in vivo obtaining of a (liquid) sample fromsuch a human or animal body is not an essential part of the methods ofthe invention described herein. In particular, the obtaining of a liquidsample can take place before practice of, and so be omitted from, themethods according to the fifth to ninth aspects of the present inventionsuch that the liquid sample, or sample of interest (also typically aliquid sample) may be defined as having been previously obtained from aliving being. Alternatively the methods according to the fifth to ninthaspects of the invention may specifically not be a diagnostic methodpractised on the human or animal body.

In this way the methods according to the seventh to ninth aspects of thepresent invention may be, or be part of, an in vitro diagnostic method.

Excitation of lanthanide (III) complexes of the invention in solutionwith wavelengths in the range 335-405(±20) nm afford metal-basedemission spectra with emission bands from about 580 to about 700 nm. Asis known (P. Atkinson et al., Org. Biomol. Chem., 2006, 4, 1707)coordination of the pyridyl nitrogen atom of aza(thia)xanthones tolanthanide (III) ions permits efficient sensitisation of lanthanideemission with the λ_(exc) varying with the particular aza(thia)xanthoneemployed. For example complexes comprising compounds of formula (Ia) areappropriately excited with radiation of about 336 or 337 (±20) nm;complexes comprising compounds of formula (Ib) are appropriately excitedwith radiation of about 380 (±25) nm. In each case, the most appropriateexcitation energy can be selected by the skilled person, by reference tothe resultant emission spectrum.

Preparatory to practising the various methods of this invention, acalibration curve may be established in order to allow correlationbetween emission bands with known quantities of citrate and lactate in arelevant calibration sample. Such preparatory methods allow appropriateworking ranges for the analysis to be defined.

For example, as described in greater detail below, a simulated prostatefluid background containing 0.3 M HSA, 0.1 NaCl, 4 mM KCl, 3 mM NaHCO₃ 4mM CaCl₂, 2 mM ZnCl₂, 5 mM MgCl₂ (pH 6.5, 0.1 M HEPES) allows modulationof europium emission at 10 μM concentration) with lactate concentrationsin the range 0 to 1 mM.

It may be desirable to dilute liquid samples obtained clinically, e.g.seminal or prostatic fluid samples) prior to practice of the methods ofthis invention in order that typical lactate or citrate concentrationsfound in clinical sample can be diluted into the working concentrationsof the analysis. Appropriate dilution protocols can be establishedeasily by those skilled in the art. For example, a 1 μL as a sample offluid (e.g. seminal or prostate) may be conveniently diluted by a factorof 100 allowing analysis in a 50 μL optical cuvette.

Also preparatory to practice of the various methods of this inventionmay be filtration of the samples obtained initially (e.g. clinically),for example to remove large biomolecules such as proteins orpolysaccharides, which might otherwise interfere with the analysis. Thismay easily be achieved by filtration, for example, through a 10 kDcut-off filter. Using these methods, as is described in greater detailin the experimental section, lactate samples in urine (3.5 simple mM),seminal fluid (3.8 mM), prostate fluid (4 mM) and reconstituted humanserum (1.9 mM) were found to be within 10% of the concentrationdetermined enzymatically. In saliva, each method (enzymatic and of theinvention) gave a zero reading for lactate (±) 0.2 mM).

Analogously, preparatory methods (including dilution and filtration) canbe undertaken in connection with the analysis of citrate. Typically,prostatic fluids are diluted by a factor of about 100 prior to analysis.According to the methods of the invention as described in greater detailhereinbelow, citrate concentrations ranging from 12 mM to 160 mM in 14samples of prostate fluid were measured and confirmed to be within 10%of the concentrations deduced using a citrate lyase enzyme kit (forwhich the amount of bio-fluid required was notably 25 times greater).Using similar methods, but with a 10-fold dilution, citrate could bedetermined in the urine of healthy volunteers between a range of from3.5-5.5 mM (±10%).

In accordance with the methods of this invention, after a contact of asample of interest with a complex of the present invention, andoptionally the preparatory steps described herein, analysis for thepresence of citrate or lactate may be achieved by analysis of thespectrum of luminescent radiation emitted upon excitation for anymodulation resultant from contact with the sample of interest. Whilst itwill be appreciated that the modulation in intensity of any givenemission band within the emission spectrum may be measured to reportupon the quantity or concentration of the analyte under analysis, theprinciple of ratiometric detection, in which changes in the ratioemissivities at two different wavelengths are compared, as is known tothose in the art, imparts a greater precision to the analysis. Inparticular, ratiometric assays typically allow a 3% variance in themeasured intensity ratio for a given concentration of analyte (citrateor lactate).

As described below, ratiometric detection of citrate in samples ofinterest may be achieved by monitoring ratios between up to sixdifferent emission wavelengths as a function of citric acidconcentration. With a complex of the present invention of formula (Ia),the intensity ratios of the 613/586 or 614/683 nm emission bands may bemeasured (although it is again stressed that it will be appreciated bythose skilled in the art that the particular emission bands and theirλ_(max) will vary depending upon the particular complexes used).

In the analysis of lactate in samples of interest using a complex of theinvention, as described below, ratiometric detection may be achieved byplotting changes in the ratio of up to 4 different wavelengths. In thisway, the intensity ratio of the 692/619, 613/622, or 613/619 nm emissionbands may be measured against change in lactate concentration whereby toestablish a calibration curve.

Thus, it will be appreciated that, by using known quantities of citrateor lactate, it is possible to construct calibration curves that may beused to determine the presence, and if so the quantity or concentration,of citrate or lactate present in samples of interest. It will also beappreciated that dilution of the sample of interest may be necessary oradvantageous prior to contact with the complex of the invention inaccordance with the working range of detection established fromconstruction of the calibration curve. The construction of appropriatecalibration curves, working ranges for citrate and lactateconcentrations and appropriate dilution (or concentration) modificationsare well within the abilities of those skilled in the art.

The use of time-gated measurements may be used to eliminate anyinterference from concomitant chromophore fluorescence, in particular inthe analysis of citrate according to the methods of this invention.

It will immediately be understood by those skilled in the art how themethods of the invention may be used and are useful in the screening ordiagnosis of the various medical conditions related to abnormalconcentrations of citrate or lactate found in various bodily samples, inallowing a determination of the concentrations of these analyses insubjects. For example, the medical practitioner will be able tocorrelate the concentration of citrate, or lactate, found in samples ofinterest, e.g. of clinical origin, with typical concentrations found inhealthy patients, or in patients susceptible to, or suffering frommedical conditions such as prostate cancer, associated with excessivelactic acid (e.g. lactic acidosis, hyperlactemia) or renal abnormalitiessuch as renal metabolic imbalance, nephrocalcinosis or nephrolithiasis.

In summary, the invention provides for a rapid luminescence measurementthat allows the determination of citrate or lactate in low volume (<5μL) samples of various biological fluids. The methods are rapid (<5min.) and offer considerable promise as alternatives to classicalenzymatic assays.

The mention of all patent of other publications referred to herein is tobe understood as if each and every one of these publications had beenspecifically incorporated by reference in their entirety.

The invention is now illustrated by the following non-limiting examples:

EXAMPLE 1 Synthesis of [Eu.L⁸]

1,7-Bis(α-dimthylglutarate)-4-[(1-azaxanthone)-2-methyl]-1,4,7,10-tetraaza-cyclododecane

1,7-Bis(α-dimethylglutarate)-1,4,7,10-tetraazacyclododecane (130 mg, 266μmol) was combined with 2-bromomethyl-1-azaaxanthone (1 eq., 80 mg) andNaHCO₃ (1.1 eq., 24 mg) and the mixture stirred in dry MeCN (4 mL) at55° C. under argon for 48 h. The reaction was monitored by TLC(DCM:MeOH, 96:4) and ESMS⁺ to confirm that the brominated startingmaterial had been consumed. The solvent was removed under reducedpressure and the resulting solid was dissolved in a small volume of DCM(5 mL) and the sodium salts filtered off. The crude mixture was purifiedby column chromatography (DCM→0.2% MeOH) to yield the title compound asa yellow oil (82 mg, 118 μmol, 44%); δ_(H) (CDCl₃) 8.64 (1H, d, J 8.0Hz, H⁴), 8.27 (1H, d, J 8.0 Hz, H⁶), 7.89 (1H, d, J 8.0 Hz, H⁹), 7.78(1H, t, J 8.0 Hz, H⁸), 7.40 (1H, t, J 8.0 Hz, H²), 7.31 (1H, d, J8.0 Hz,H³), 3.88 (2H, s, H¹⁰), 3.67 (6H, s, H¹⁶), 3.57 (6H, s, H¹²), 3.35 (2H,t, H¹³), 3.00 (16H, m, H^(11,11′,12,12′)), 2.36 (4H, m, H¹⁵), 1.93 (4H,m, H¹⁴); δ_(c) (CDCl₃) 177.6 (C⁵) 173.3 (C^(16′)), 172.6 (C^(17′)),162.9 (C²), 160.3 (C^(4′)), 155.9 (C^(6′)), 138.1 (C⁴), 136.1 (C⁸),126.7 (C⁶), 125.0 (C⁷), 121.7 (C^(1′)), 121.3 (C³), 119.3 (C⁹), 115.9(C^(9′)), 65.5 (C¹³), 53.7 (C¹⁰), 51.9 (C^(16,17)), 51.4, 50.8, 48.9,46.7 (C^(11,11′,12,12′)), 30.8 (C¹⁵), 25.7 (C¹⁴); R_(f) 0.38 (DCM−4%MeOH, alumina); m/z (HRMS⁺) 698.3403 (M+H)⁺ (C₃₅H₄₈O₁₀N₅ requires689.3396).

1,7-Bis(α-glutarate)-4-[(1-azaxanthone)-2-methyl]-1,4,7,10-tetraaza-cyclododecane

Freshly made aqueous KOD solution (3 mL, 0.1 M) was added to1,7-bis(α-dimethylglutarate)-4-[(1-azaxanthone)-2-methyl]-1,4,7,10-tetraaza-cyclododecane(42 mg, 61 μmol). The reaction mixture was kept under argon at roomtemperature and progress was monitored by NMR. After 6h no methyl estersignals were observed in the ¹H-NMR spectrum. The pH of the mixture wasdecreased (pH≈6) with conc. HCl and the solution loaded onto a DOWEX50X4-100 strong cation exchange resin. The column was eluted withwater→10% NH₄OH and the fractions were analysed by ^(1H)-NMR. Thefractions were combined and lyophilised to yield the title compound as apale yellow glass (15 mg, 23.5 μmol, 39%), which was used in acomplexation reaction immediately. δ_(H) (D₂O) 8.56 (1H, d, J8.0 Hz,H⁴), 8.02 (1H, d, J8.0 Hz, H⁶), 7.78 (1H, d, J 8.0 Hz, H⁹), 7.41 (3H,br.m, H^(8,3,7)), 3.53 (2H, s, H¹⁰), 3.09 (18H, br.m,H^(11,11′,12,12′,13)), 2.11 (8H, br.m, H^(14,15)); m/z (ESMS⁻) 637(M−H)⁻.

[NaEuL⁸]

1,7-Bis(α-glutarate)-4-[(1-azathiaxanthone)-2-methyl]-1,4,7,10-tetraaza-cyclododecane(15 mg, 23.5 μmol) was added to Eu(CH₃CO₂)₃.3H₂O (1.1 eq., 10 mg) andthe solids dissolved in aqueous methanol (10:1, 3 mL). The pH wascarefully adjusted to 5.5 by addition of acetic acid and the reactionleft to stir at 70° C. for 48 hrs. After the reaction was cooled to roomtemperature, the solvents were removed under reduced pressure and theremaining residue was dissolved in H₂O (3 mL). The pH was adjustedcarefully to 10 by addition of conc. aqueous NaOH solution (in order toremove any excess Eu³⁺ as Eu(OH)₃) resulting in a white precipitate thatwas removed via centrifugation. The pH was adjusted back to neutral withacetic acid and the mixture lyophilised to give a bright yellow solidcontained about 2% NaOAc as a result of pH adjustment (14 mg, 20 μmol).m/z (HRMS⁻) 791.1646 (M−H)⁻ (C₃₁H₃₅O₁₀N₅ ¹⁵¹Eu requires 791.1635);λ_(max)(H₂O) 336 (5010 dm³mol⁻¹cm⁻¹) τ^(Eu) _((H2O, pH=3.0)): 0.37 ms,τ^(Eu) _((H2O, pH=8.0)): 0.41 ms; τ^(Eu) _((D2O,pD=2.6)): 0.93 ms,τ^(Eu) _((D2O,pD=7.6)): 0.98 ms; φ^(Eu) _((pH=3.0))=8%, φ^(Eu)_((pH=8.0))=5%.

EXAMPLE 2 Synthesis of [Eu.L⁹]

1,7-Bis(α-dimethylglutarate)-4-[(1-azaxanthone)-2-methyl]-10-methyl-1,4,7,10-tetraaza-cyclododecane

1,7-Bis(α-dimethylglutarate)-4-[(1-azaxanthone)-2-methyl]-1,4,7,10-tetraaza-cyclododecane(40 mg, 58 μmol) was added to a solution of formic acid (1 mL) andaqueous formaldehyde (38%, 1 mL) and the mixture was boiled under refluxfor 48 h. After filtration and removal of the solvent, the residue wastreated with aqueous sodium hydroxide solution (2 M, 5 mL) and extractedwith chloroform (3×10 mL). The combined extracts were dried and thesolvent was removed under reduced pressure to yield a bright yellowsolid (40 mg, 56 μmol, 97%); δ_(H) (CDCl₃) 8.64 (1H, d, J 8.0 Hz, H⁴),8.25 (1H, d, J 8.0 Hz, H⁶), 7.86 (1H, m, H⁹), 7.79 (1H, t, J 8.0 Hz,H⁸), 7.34 (2H, m, J 8.0 Hz, H^(3,7)), 3.88 (2H, s, H¹⁰), 3.67 (6H, s,H¹⁶), 3.57 (6H, s, H¹⁷), 3.35 (2H, t, H¹³), 3.00 (21H, m,H^(11,11′,12,12′,18(dist. s at 2.94))), 2.34 (4H, m, H¹⁵), 1.90 (4H, m,H¹⁴); m/z (HRMS⁺) 712.3564 (M+H)⁺ (C₃₆H₅₀O₁₀N₅ requires 712.3552).

1,7-Bis(α-glutarate)-4-[(1-azaxanthone)-2-methyl]-10-methyl-1,4,7,10-tetraaza-cyclododecane

Freshly made aqueous KOD solution (2.5 mL, 0.1 M) was added to1,7-bis(α-dimethylglutarate)-4-[(1-azathiaxanthone)-2-methyl]-10-methyl-1,4,7,10-tetraaza-cyclododecane(40 mg, 56 μmol). The reaction mixture was kept under argon at roomtemperature and progress was monitored by NMR. After 4 h no methyl esterresonances were observed in the ¹H-NMR spectrum. The pH of the mixturewas decreased (pH≈6) with conc. HCl and the solution loaded onto a DOWEX50X4-100 strong cation exchange resin. The column was eluted withwater→10% NH₄OH and the fractions were analysed by 1H-NMR. The fractionswere combined and lyophilised to yield the title compound as a brightyellow powder (22 mg, 34 μmol, 61%), which was used in a complexationreaction immediately. δ_(H) (D₂O): mainly broad overlapping signals; noMe groups in ¹H-NMR, δ_(H) (D₂O) 8.74 (1H, d, J 8.0 Hz, H⁴), 8.17 (1H,d, J 8.0 Hz, H⁶), 7.90 (2H, br.m, H^(8,9)), 7.57 (2H, br.m, J 8.0 Hz,H^(3,7)), 3.80 (2H, s, H¹⁰), 3.27 (2H, t, H₁₃), 2.96 (21H, br.m,H^(11,11′,12,12′,18(dist. s at 2.86))), 2.44 (4H, br.m, H¹⁵), 1.97 (4H,br.m, H¹⁴); m/z (ESMS⁻) 651 (M−H)⁻.

[NaEuL⁹]

1,7-Bis(α-glutarate)-4-[(1-azathiaxanthone)-2-methyl]-10-methyl-1,4,7,10-tetraaza-cyclododecane(22 mg, 34 μmol) was added to Eu(CH₃CO₂)₃.3H₂O (1.1 eq., 15 mg) and thesolids dissolved in aqueous methanol (10:1 mL). The pH was carefullyadjusted to 5 by addition of acetic acid and the reaction left to stirat 55° C. for 30 h. After the reaction was cooled to room temperature,the solvents were removed under reduced pressure and the remainingresidue was dissolved in H₂O (5 mL). The pH was then adjusted carefullyto 10 by addition of conc. aqueous NaOH solution (in order to removeexcess Eu³⁺ as Eu(OH)₃) resulting in a white precipitate that wasremoved via centrifugation. The pH was adjusted back to neutral withacetic acid and the mixture lyophilised to give a bright yellow solidcontained about 2% NaOAc as a result of pH adjustment (22 mg, 27 μmol).m/z (HRMS⁻) 804.1759 (M−H)⁻ (C₃₂H₃₈O₁₀N₅ ¹⁵¹Eu requires 804.1758);λ_(max)(H₂O) 336 (5010 dm³mol⁻¹cm⁻¹), τ^(Eu) _((H2O, pH=3.0)): 0.34 ms,τ^(Eu) _((H2O, pH=8.0)): 0.77 ms; τ^(Eu) _((D2O,pD=2.6)): 1.27 ms,τ^(Eu) _((D2O,pD=7.6)): 1.26 ms; φ^(Eu) _((pH=3.0))=26%, φ^(Eu)_((pH=8.0))=11%

EXAMPLE 3 Synthesis of [Eu.L⁵] (Comparative Example)

(SS)-1,7-Bis(ethoxycarbonyl-2-ethylcarbomoylmethyl)-4-[(1-azaxanthone)-2-methyl]-1,4,7,10-tetraazacyclododecane

(SS)-1,7-Bis(ethoxycarbonyl-2-ethylcarbamoylmethyl)-1,4,7,10-tetraazacyclododecane(590 mg, 1.22 mmol) was combined with 2-bromomethyl-1-azaxanthone (1eq., 350 mg) and NaHCO₃ (1 eq., 102 mg) and the mixture in dry MeCN (25mL), was heated at 55° C. for 20 h under argon. The reaction wasmonitored by TLC (DCM, 3.5% MeOH, alumina) and ESMS⁺ to confirm that thebrominated starting material had been consumed. The solvent was removedunder reduced pressure and the resulting solid was dissolved in a smallvolume of DCM (5 mL) and the potassium salts removed by filtration. Thecrude mixture was purified by column chromatography (DCM→3% MeOH,alumina); fractions containing clean product were combined and thesolvents were removed under reduced pressure to yield the title compoundas a pale yellow oil (300 mg, 432 μmol, 36%); δ_(H) (CDCl₃): 8.61 (H⁴,d, 1H, J=8.0 Hz), 8.25 (H⁶, dd, 1H, J=8.0 Hz), 7.76 (H⁸, dt, 1H, J=8.0Hz), 7.61 (H⁹, d, 1H, J=8.0 Hz), 7.48 (H¹⁵, br. s, 2H), 7.38 (H^(3,7),m, 2H), 4.40 (H¹⁶, 2H, p, J=7.0 Hz), 4.08 (H¹⁹, q, 4H, J=7.0 Hz), 3.98(H¹⁰, s, 2H), 3.20 (H^(11,11′,12,12′,13), m, 20H) 1.34 (H¹⁷, d, 6H,J=7.0 Hz), 1.19 (H²⁰, t, 6H, J=7.0 Hz); δ_(c) (CDCl₃) 177.4 (C⁵), 173.2(C¹⁸), 170.4 (C¹⁴), 164.0 (C²), 160.2 (C^(1′)), 155.8 (C^(9′)), 138.1(C⁴), 136.0 (C⁸), 126.9 (C⁶), 125.1 (C⁷), 121.8 (C^(6′)), 121.4 (C³),118.7 (C⁹), 115.8 (C^(4′)), 61.5 (C¹⁹), 60.3 (C¹⁰), 56.2 (C¹³), 54.1,52.9, 51.4, 47.1 (C^(11,11′,12,12′)), 47.1 (C¹⁶), 17.9 (C¹⁷), 14.3(C²⁰), m/z (HRMS⁺) 696.3712 (M+H)⁺ (C₃₅H₅₀O₈N₇ requires 696.3715) R_(f)0.40 (alumina, DCM with 3.5% MeOH).

(SS)-1,7-Bis(carboxy-2-ethylcarbamoylmethyl)-4-[(1-azaxanthone)-2-methyl]-1,4,7,10-tetraazacyclododecane

Freshly made aqueous KOD solution (5 mL, 0.1 M) was added to(SS)-1,7-bis(ethoxycarbonyl-2-ethylcarbamoylmethyl)-4-[(1-azaxanthone)-2-methyl]-1,4,7,10-tetraazacyclo-dodecane(85 mg, 122 μmol). The reaction mixture was kept under argon at roomtemperature and progress was monitored by NMR. After 6 h no ethyl groupsignals were observed in the ¹H-NMR spectrum. The pH of the mixture wasincreased (pH≈6) with conc. HCl and the solution loaded onto a DOWEX50X4-100 strong cation exchange resin. The column was eluted withwater→10% NH₄OH and the fractions analysed by ¹H-NMR. Selected fractionswere combined and lyophilised to yield the title compound as a paleyellow oil (30 mg, 47 μmol, 39%), which was used for complexationreaction immediately. δ_(H) (D₂O): 8.23 (H⁴, br.d, 1H, J=7.8 Hz), 7.83(H⁶, br.d, 1H, J=7.8 Hz), 7.66 (H⁸,br.t, 1H, J=7.8 Hz), 7.25 (H^(3,7,9),br.m, 3H, J=7.8 Hz), 4.08 (H¹⁶, br.m, 2H), 3.09 (H¹⁰, s, 2H), 3.02(H^(11,11′,12,12′,13), br.m, 22H), 0.87 (H¹⁷, d, 6H, J=7.1 Hz)

[EuL⁵]OAc

(SS)-1,7-Bis(carboxy-2-ethylcarbamoylmethyl)-4-[(1-azaxanthone)-2-methyl]-1,4,7,10-tetraazacyclododecane(30 mg, 47 μmol) was added to Eu(OAc)₃.4H₂O (1.1 eq., 19 mg) and thesolids dissolved in aqueous methanol (10:1, 2.5 mL). The pH wascarefully adjusted to 6 by addition of acetic acid and the reaction leftto stir at 75° C. for 60 h. After the reaction was cooled to roomtemperature, the pH was adjusted carefully to 10 by addition of aqueousammonia solution (35%) (in order to remove excess Eu as Eu(OH)₃)resulting in a white precipitate that was removed via centrifugation.The pH was adjusted back to neutral with acetic acid and the samplelyophilised to give a bright yellow solid (25 mg, 32 μmol, 68%). m/z(HRMS⁺) 790.2121 (M+H) (C₃₁H₃₉O₇N₇Eu requires 790.2070); λ_(max)(H₂O)336 (5010 dm³mol⁻¹cm⁻¹), τ^(Eu) _((H2O, pH=6.0)): 0.39 ms, τ^(Eu)_((D2O, pD=6.0)): 0.92 ms; φ^(Eu) _((pH=6.0))=5%

EXAMPLE 4 Synthesis of [Eu.L⁶] (Comparative Example)(SS)-1,7-Bis(ethoxycarbonyl-2-ethylcarbamoylmethyl)-4-[(1-azaxanthone)-2-methyl]-10-methyl-1,4,7,10-tetraazacyclododecane

(SS)-1,7-Bis(ethoxycarbonyl-2-ethylcarbamoylmethyl)-4-[(1-azaxanthone)-2-methyl]-1,4,7,10-tetraazacyclododecane(100 mg, 144 μmol) was added to a solution of formic acid (2 mL) andaqueous formaldehyde (38%, 2 mL) and the mixture was boiled under refluxfor 20 h. After removal of the solvent under reduced pressure, theresidue was treated with DCM:MeOH (10 mL, 10:1) and the paraformaldehydeby-product was removed via a combination of filtration andcentrifugation. The organic solvent was removed under reduced pressureto yield the title compound as a bright yellow solid (84 mg, 118 μmol,83%); δ_(H) (CDCl₃): 8.63 (H⁴, d, 1H, J=7.9 Hz), 8.23 (H⁶, dd, 1H, J=7.9Hz), 7.75 (H⁸, dt, 1H, J=7.9 Hz), 7.54 (H^(3,9), m, 2H, J=7.9 Hz), 7.38(H⁷, dt, 1H, J=7.9 Hz), 6.78 (H¹⁵, br. s, 2H), 4.36 (H¹⁶, 2H, p, J=7.0Hz), 4.06 (H^(10,19), m, 6H), 3.15(H^(11,11′,12,12′,13,21 (dist. s at 2.97)), m, 20H), 1.35 (H¹⁷, d, 6H,J=7.0 Hz), 1.18 (H²⁰, t, 6H, J=7.0 Hz); δ_(c) (CDCl₃) 177.3 (C⁵), 172.8(C¹⁸), 170.8 (C¹⁴), 165.2 (C²), 160.1 (C^(1′)), 155.7 (C^(9′)), 138.6(C⁴), 136.1 (C⁸), 126.9 (C⁶), 125.2 (C⁷), 121.8 (C^(6′)), 121.7 (C³),118.6 (C⁹), 116.0 (C^(4′)), 61.5 (C¹⁹), 60.6 (C¹³), 56.0 (C¹⁰), 53.8,53.5, 49.9, 47.4 (C^(11,11′,12,12′)), 48.5 (C¹⁶), 43.7 (C²¹),17.3 (C¹⁷),14.3 (C²⁰), m/z (HRMS⁺) 710.3870 (M+H)⁺ (C₃₆H₅₂O₈N₇ requires 710.3857)

(SS)-1,7-Bis(carboxy-2-ethylcarbamoylmethyl)-4-[(1-azaxanthone)-2-methyl]-10-methyl-1,4,7,10-tetraazacyclododecane

Freshly made aqueous KOD solution (5 mL, 0.1 M) was added to(SS)-1,7-bis(ethoxycarbonyl-2-ethylcarbamoylmethyl)-4-[(1-azaxanthone)-2-methyl]-10-methyl-1,4,7,10-tetraazacyclo-dodecane(84 mg, 118 μmol). The reaction mixture was kept under argon at roomtemperature and progress was monitored by NMR. After 6 h no ethyl groupsignals were observed in the ¹H-NMR spectrum. The pH of the mixture wasincreased (pH≈6) with conc. HCl and the solution loaded onto a DOWEX50X4-100 strong cation exchange resin. The column was eluted withwater→10% NH₄OH and the fractions were analysed by ¹H-NMR. The fractionswere combined and lyophilised to yield the title compound as a paleyellow oil (30 mg, 46 μmol, 39%), which was used in a complexationreaction immediately. δ_(H) (D₂O): 8.18 (H⁴, br.d, 1H, J=8.0 Hz), 7.74(H^(6,8), br.m, 2H, J=8.0 Hz), 7.21 (H^(3,7,9), br.m, 3H, J=8.0 Hz),3.84 (H¹⁶, br.m, 2H), 3.15 (H₁₀, s, 2H), 3.02(H^(11,11′,12,12′,13,21 (dist. s at 2.78)), br.m, 25H), 1.05 (H¹⁷, d,6H, J=7.2 Hz)

[EuL⁶]OAc

(SS)-1,7-Bis(carboxy-2-ethylcarbamoylmethyl)-4-[(1-azaxanthone)-2-methyl]-10-methyl1,4,7,10-tetraazacyclododecane (30 mg, 47 μmol) was added toEu(OAc)₃.4H₂O (1.1 eq., 19 mg) and the solids dissolved in 2.5 mLH₂O:MeOH (10:1). The pH was carefully adjusted to 6 by addition ofacetic acid and the reaction left to stir at 75° C. for 60 h. After thereaction was cooled to room temperature, The pH was then adjustedcarefully to 10 by addition of aqueous ammonia (35%) solution (in orderto remove any excess Eu as Eu(OH)₃) resulting in a white precipitatethat was removed via centrifugation. The pH was adjusted back to neutralwith acetic acid and the sample lyophilised to give a bright yellowsolid (23 mg, 29 μmol, 61%). m/z (HRMS⁺) 804.2232 (M+H) (C₃₂H₄₁O₇N₇Eurequires 804.2230); λ_(max)(H₂O) 336 (5010 dm³mol⁻¹cm⁻¹); τ^(Eu)_((H2O, pH=6.0)): 0.62 ms, τ^(Eu) _((D2O, pD=6.0)): 1.42 ms; φ^(Eu)_((pH=6.0))=5%.

EXAMPLE 5 Synthesis of [Eu.L¹] (Comparative Example)(SS)-1,7-Bis(ethoxycarbonyl-2ethylcarbamoylmethyl)-4-[(1-azathiaxanthone)-2-methyl]-1,4,7,10-tetraazacyclododecane

(SS)-1,7-Bis(ethoxycarbonyl-2-ethylcarbamoylmethyl)-1,4,7,10-tetraazacyclododecane(200 mg, 411 μmol) was combined with 2-bromomethyl-1-azathiaxanthone (1eq., 126 mg) and KHCO₃ (1 eq., 41 mg) and the mixture stirred in dryMeCN (10 mL), was heated at 70° C. for 36 h under argon. The reactionwas monitored by TLC (DCM, 5% MeOH, alumina) and ESMS⁺ to confirm thatthe brominated starting material had been consumed. The solvent wasremoved under reduced pressure and the resulting solid was dissolved ina small volume of DCM (5 mL) and the potassium salts removed byfiltration. The crude mixture was purified by column chromatography(DCM→3% MeOH, alumina); fractions containing clean product were combinedand the solvents were removed under reduced pressure to yield the titlecompound as a pale yellow oil (130 mg, 182 μmol, 45%); δ_(H) (CDCl₃):8.74 (H⁴, d, 1H, J=8.1 Hz), 8.57 (H⁶, d, 1H, J=8.2 Hz), 7.66(H^(8,9,21), m, 3H), 7.53 (H⁷, t, 1H, J=8.2 Hz), 7.39 (H³, d, 1H, J=8.1Hz), 7.33 (H¹⁵, br.s, 2H), 4.51 (H¹⁶, 2H, p, J=7.1 Hz), 4.13 (H¹⁹, q,4H, J=7.1 Hz), 3.93 (H¹⁰, m, 2H), 3.15 (H^(11,11′,12,12′,13), m, 20H)1.41 (H¹⁷, t, 4H, J=7.1 Hz), 1.24 (H²⁰, q, 6H, J=7.1 Hz); δ_(c) (CDCl₃)180.5 (C⁵), 173.3 (C¹⁸), 170.4 (C¹⁴), 162.4 (C²), 158.6 (C^(1′)), 138.7(C⁴), 137.0 (C^(4′)), 133.4 (C⁹), 130.2 (C⁶), 129.1 (C^(6′)), 127.3(C⁷), 126.8 (C⁸), 125.5 (C^(9′)), 122.4 (C³), 61.7 (C¹⁹), 60.6 (C¹⁰),56.0 (C¹³), 54.2, 53.7, 53.0, 47.0 (C^(11,11′,12,12′)), 48.4 (C¹⁶), 18.2(C¹⁸), 14.4 (C²⁰), m/z (HRMS⁺) 712.3484 (M+H)⁻ (C₃₅H₅₀O₇N₇S requires712.3487) R_(f) 0.36 (alumina, DCM−5% MeOH).

[EuL¹](CF₃SO₃)₃

(SS)-1,7-Bis(ethoxycarbonyl-2-ethylcarbamoylmethyl)-4-[(1-azathiaxanthone)-2-methyl]-1,4,7,10-tetraazacyclododecane(18 mg, 25 μmol) was added to Eu(CF₃SO₃)₃ (1 eq., 16 mg) and the solidsdissolved in dry MeCN (3 mL) and the reaction left to stir at 80° C. for74 h. After the reaction was cooled to room temperature, the solventswere removed under reduced pressure and the remaining residue wasdissolved in 0.1 mL dry MeCN and the mixture was dropped onto anhydrousEt₂O which resulted in precipitation of the title compound as a triflatesalt. The precipitate was spinned out and dissolved in 5 ml H₂O:MeOH(3:1) The pH was then adjusted carefully to 10 by addition of conc. NaOHsolution (in order to remove the excess Eu as Eu(OH)₃) resulting in awhite precipitate, whish was removed by centrifugation. The pH wasadjusted back to neutral with aqueous HCL and the mixture lyophilised togive a bright yellow solid contained aprox 2% NaCl as a result of pHadjustment (16 mg, 11.5 μmol). m/z (HRMS⁺) 1162.1618 (M+2CF₃SO₃)⁺(C₃₅H₄₉O₇N₇SEu(CF₃S0₃)₂ requires 1162.1667); λ_(max)(H₂O) 380 (4070dm³mol⁻¹cm⁻¹); τ^(Eu) _((H2O, pH=7.4)): 0.24 ms, τ^(Eu)_((D2O, pD=7.1)): 0.56 ms; φ^(Eu) _((pH=7.4))=4.4%

EXAMPLE 6 Synthesis of [Eu.L³](SS)-1,7-Bis(carboxy-2-ethylcarbamoylmethyl)-4-[(1-azathiaxanthone)-2-methyl]-1,4,7,10-tetraazacyclododecane

Freshly made aqueous KOD solution (2.5 mL, 0.1 M) was added to(SS)-1,7-bis(ethoxycarbonyl-2-ethylcarbamoylmethyl)-4-[(1-azathiaxanthone)-2-methyl]-1,4,7,10-tetraazacyclododecane(61 mg, 86 μmol). The reaction mixture was kept under argon at roomtemperature and progress was monitored by NMR. After 6 h no ethyl groupsignals were observed in the ¹H-NMR spectrum. The pH of the mixture wasincreased (pH≈6) with conc. HCl and the solution loaded onto a DOWEX50X4-100 strong cation exchange resin. The column was eluted withwater→10% NH₄OH and the fractions were analysed by ¹H-NMR. The fractionswere combined and lyophilised to yield the title compound as a paleorange oil (43 mg, 66 μmol, 77%), which was used for complexationimmediately. δ_(H) (D₂O): 8.17 (H⁴, br.d, 1H), 7.98 (H⁶, br.d, 1H), 7.47(H⁷,br.t, 1H), 7.25 (H^(8,9,3,15,21), m, 7H), 4.02 (H¹⁶, m, 2H, J=8.0Hz), 3.73 (H¹⁰, br.s, 2H), 3.10 (H^(111,11′,12,12′,13), m, 20H), 1.14(H¹⁷, d, 6H, J=7.5 Hz).

[EuL³(OAc)]

(SS)-1,7-Bis(carboxy-2-ethylcarbamoylmethyl)-4-[(1-azathiaxanthone)-2-methyl]-1,4,7,10-tetraazacyclododecane(43 mg, 66 μmol) was added to Eu(OAc)₃.4H₂O (1.2 eq., 33 mg) and thesolids dissolved in 2.5 mL H₂O:MeOH (5:1). The pH was carefully adjustedto 5 by addition of acetic acid and the reaction left to stir at 75° C.for 60 h. After the reaction was cooled to room temperature, The pH wasthen adjusted carefully to 10 by addition of conc. NaOH solution (inorder to remove excess europium as Eu(OH)₃) resulting in a whiteprecipitate removed via centrifugation. The pH was adjusted back toneutral with acetic acid and the sample lyophilised to give a brightyellow solid contained about 2% NaOAc as a result of pH adjustment (20mg, 22 μmol). m/z (HRMS⁺) 890.2004 (M+Na+OAc) (C₃₁H₄₁O₂N₂SEuNaCH₃COOrequires 890.2031); λ_(max)(H₂O) 380 (4070 dm³mol⁻¹cm⁻¹); τ^(Eu)_((H2O, pH=7.2)): 0.24 ms, τ^(Eu) _((D2O, pD=7.0)): 0.70 ms; φ^(Eu)_((pH=7.4))=1.2%

EXAMPLE 7 Synthesis of [Eu.L⁴](SS)-1,7-Bis(carboxy-2-ethylcarbamoylphenyl)-4-[(1-azathiaxanthone)-2-methyl]-1,4,7,10-tetraazacyclododecane

Freshly made aqueous KOD solution (2.5 mL, 0.1 M) was added to(SS)-1,7-bis(ethoxycarbonyl-2-ethylcarbamoylphenyl)-4-[(1-azathiaxanthone)-2-methyl]-1,4,7,10-tetraazacyclododecane(51 mg, 59 μmol) with 0.3 mL CD₃OD. The reaction mixture was kept underargon at room temperature and progress was monitored by NMR. After 10 hethyl group signals were observed in the ¹H-NMR spectrum. The pH of themixture was increased (pH≈6) with conc. HCl and the solution loaded ontoa DOWEX 50X4-100 strong cation exchange resin. The column was elutedwith water→10% NH₄OH and the fractions were analysed by ¹H-NMR. Thefractions were combined and lyophilised to yield the title compound as apale orange oil (25 mg, 32 μmol, 54%), which was used for complexationreaction immediately. δ_(H) (D₂O) 8.22 (H⁴, br.s., 1H), 7.94 (H⁶, br.s.,1H), 7.00 (H^(3,7,8,9,15,18,21), m, 17H), 4.63 (H¹⁶, m, 2H), 4.22(H^(10,20), m, 6H), 2.76 (H^(11,11′,12,12′,13,17), m, 24H)

[EuL⁴](OAc)

(SS)-1,7-Bis(carboxy-2-ethylcarbamoylphenyl)-4-[(1-azathiaxanthone)-2-methyl]-1,4,7,10-tetraazacyclododecane(25 mg, 32 μmol) was added to Eu(OAc)₃.4H₂O (1.1 eq., 16 mg) and thesolids dissolved in 2.5 mL H₂O:MeOH (5:1). The pH was carefully adjustedto 5 by addition of acetic acid and the reaction left to stir at 75° C.for 64 h. After the reaction was cooled to room temperature, The pH wasthen adjusted carefully to 10 by addition of conc. aqueous NaOH solution(in order to remove excess europium as Eu(OH)₃) resulting in a whiteprecipitate removed via centrifugation. The pH was adjusted back toneutral with acetic acid and the sample lyophilised to give a brightyellow solid contained about 2% NaOAc as a result of pH adjustment (20mg, 22 μmol). m/z (HRMS⁻) 1034.2991 (M+Me+OAc) (C₄₃H₄₉O₇N₇SEuCH₃COO (asa mono Me-ester) requires 1034.2994); λ_(max)(H₂O) 380 (4070dm³mol⁻¹cm⁻¹); τ^(Eu) _((H2O, pH=7.4)): 0.24 ms, τ^(Eu)_((D2O, pD=7.0)): 0.70 ms; φ^(Eu) _((pH=7.4))=0.8%.

EXAMPLE 8 Synthesis of [Eu.L⁷] (Comparative Example)1,7-Bis(α-glutarate)-4-[(1-azathiaxanthone)-2-methyl]-1,4,7,10-tetraaza-cyclododecane

Freshly made aqueous KOD solution (2.5 mL, 0.1 M) was added to1,7-bis(α-dimethylglutarate)-4-[(1-azathiaxanthone)-2-methyl]-1,4,7,10-tetraaza-cyclododecane(70 mg, 98 μmol). The reaction mixture was kept under argon at roomtemperature and progress was monitored by NMR. After 8 h no methyl groupsignals were observed in the ¹H-NMR spectrum. The pH of the mixture wasdecreased (pH≈6) with conc. HCl and the solution loaded onto a DOWEX50X4-100 strong cation exchange resin. The column was eluted withwater→10% NH₄OH and the fractions were analysed by 1H-NMR. The fractionswere combined and lyophilised to yield the title compound as a darkyellow solid (38 mg, 57 μmol, 58%), which was used in a complexationreaction immediately. δ_(H) (D₂O): mainly broad overlapping signals; noMe groups in ¹H-NMR, δ_(H)(D₂O) 8.33 (1H, d, J 8.0 Hz, H⁴), 8.11 (1H, d,J 8.0 Hz, H⁶), 7.41 (5H, m, H^(8,9,3,7,18)), 3.53 (2H, s, H¹⁰), 3.09(18H, br.m, H^(11,11′,12,12′,13)), 1.90 (8H, m, H^(14,15)); m/z (ESMS⁻)656 (M−H)⁻.

[NaEuL⁷]

1,7-Bis(α-glutarate)-4-[(1-azathiaxanthone)-2-methyl]-1,4,7,10-tetraaza-cyclododecane(33 mg, 50 μmol) was added to Eu(CH₃CO₂)₃ (1.1 eq., 23 mg) and thesolids dissolved in a H₂O:MeOH (10:1 mL). The pH was carefully adjustedto 5 by addition of acetic acid and the reaction left to stir at 80° C.for 72 h. After the reaction was cooled to room temperature, thesolvents were removed under reduced pressure and the remaining residuewas dissolved in 5 mL H₂O. The pH was then adjusted carefully to 10 byaddition of conc. aqueous NaOH solution (in order to remove the excesseuropium as Eu(OH)₃) resulting in a white precipitate, removed viacentrifugation. The pH was adjusted back to neutral with acetic acid andthe mixture lyophilised to give a bright yellow solid contained about 2%NaOAc as a result of pH adjustment (33 mg, 37 μmol). m/z (HRMS⁻)821.1669, (M+Me)⁺ 889.1474 (M+Na+OAc)⁺ (C₃₁H₃₆O₉N₅SEu mono Me-esterrequires 821.1675, C₃₁H₃₆O₉N₅SEuNaCH₃COO requires 889.1471);λ_(max)(H₂O) 380 (4070 dm³mol⁻¹cm⁻¹); τ^(Eu) _((H2O, pH=3.0)): 0.29 ms,τ^(Eu) _((H2O, pH=8.0)): 0.30 ms; τ^(Eu) _((D2O, pD=2.6)): 0.59 ms,τ^(Eu) _((D2O, pD=7.6)): 0.63 ms; φ^(Eu) _((pH=3.0))=1.2%, φ^(Eu)_((pH=8.0))=1.2%

HPLC Analysis/Purification

Reverse phase HPLC analyses were performed at 298 K using a Perkin ElmerSystem with a 4.6×20 mm 4μ Phenomenex Synergi Fusion RP 80i analyticalcolumn. In each case an H₂O+0.1% HCOOH/MeCN+0.1% HCOOH solvent systemwas used (gradient elution) with a run time of 20 minutes. In each case,a single major product was observed in >95% purity using a diode arrayUV-Vis detector operating at 340 nm, which corresponds to the absorptionband of the appropriate azaxanthone sensitizing moiety (analysis wasalso undertaken at 280 nm). Such behaviour indicated that each of thespecies that were eluted bear this chromophore. A fluorescence detectorwas also connected to the HPLC, monitoring eluent from the column at awavelength corresponding to the Eu centred emission (616 nm); againemission was seen for each of these peaks, suggesting that each peakcorresponding to a chromophore bound species that was also coordinatedto Eu.

H₂O MeCN (+0.1% (+0.1% Time Flow HCOOH) HCOOH) (min) (mL/min) (%) (%)Gradient 3.0 1 100 0 0 (prior injection) 1.0 1 100 0 0 10.0  1 0 100 15.0 1 0 100 0 0.5 1 100 0 1 5.0 1 100 0 0 Gradient elution programme forHPLC analysis. FIGS. 1A through 1I show LC chromatograms following HPLCpurification of EuL⁸, EuL⁹, EuL¹, EuL², EuL³, EuL⁴, EuL⁷, EuL⁵ and EuL⁶.AnalysesA. Determination of Binding Constants of Complexes of the Invention andComparative Complexes

Affinity constants for lactate, citrate and bicarbonate with[Eu.L¹]³+-[Eu.L⁹]⁻ were determined in saline solution (298 K, 0.1 MNaCl, 4 mM KCl, 0.9 mM Na₂HPO₄, pH 6.55), observing the change in theintensity ratio of the Eu³⁺ emission bands at 616/686 nm as a functionof added anion, and are presented in Table 1 below. With lactate andcitrate, measurements were also made in a ‘simulated prostate fluid’background, prior to analyses in prostate fluid clinical samples. Thismedium contained various MCl₂ salts (M=Mg, Ca, Zn, C_(tot) ^(M2+)=11mM), human serum albumin (0.3 mM) and 3 mM NaHCO₃, Table 1. The addedM²⁺ salts not only compete for the oxy-anion but also appear tostabilise certain ternary [Eu.L(citrate)] adducts. For example, with[Eu.L²]³⁺, titrations of citrate (pH 7.4, 0.1 HEPES, 0.1 M NaCl, 0.9 mMNaH₂PO₄, 30 mM NaHCO₃ and 2.3 mM sodium lactate) in the absence andpresence of 2 mM MCl₂ (M=Ca, Zn and Mg) revealed an apparent affinityconstant that increased by about two log units.

TABLE 1 Comparison of apparent binding constants (log K) for europium(III) complexes with the stated anion (298K, 20 μM complex, λ_(exc) 380(azathiaxanthone) or 337 (azaxanthone) nm, pH 6.55) in salinesolution^(a) and in a simulated prostate fluid background^(b) (valuesgiven in italics), with standard deviations. Complex citrate lactatebicarbonate [Eu.L¹]³⁺ 5.26 (03) 3.31 (02) 2.81 (02) [5.20] (02) [3.41](03) [Eu.L²]³⁺ 5.22 (02) 3.27 (01) 3.07 (01) [4.88] (04) [2.97] (03)[Eu.L³]⁺ 4.58 (03) 2.94 (03) 2.55 (03) [4.51] (01) [3.20] (02) [Eu.L⁴]⁺4.01 (02) 2.79 (04) 2.23 (02) [4.20] (03) [2.82] (03) [Eu.L⁵]⁺ 4.19 (02)3.08 (02) 2.14 (02) [3.89] (03) [3.43] (05) [Eu.L⁶]⁺ 4.36 (01) 3.49 (01)2.80 (01) [3.97] (02) [3.82] (01) [Eu.L⁷]⁻ 2.52 (02) 2.46 (02) 2.11 (05)[3.12] (02) [2.90] (01) [Eu.L⁸]⁻ 2.54 (03) 3.03 (01) 1.27 (02) [1.54](03) [2.65] (02) [Eu.L⁹]⁻ 1.69 (01) 3.18 (02) 1.23 (03) [2.02] (02)[3.33] (01) ^(a)contains: 0.1M NaCl, 4 mM KCl, 0.1M HEPES and 0.9 mMNaH₂PO₄ ^(b)contains: 0.1M NaCl, 4 mM KCl, 4 mM CaCl₂, 2 mM ZnCl₂, 5 mMMgCl₂, 0.3 mM HSA, 3 mM NaHCO₃ and 0.1M HEPES.

Pronounced citrate/lactate selectivity was observed with the positivelycharged complexes, e.g. for [Eu.L³]⁺, the ratio of affinity constants is42:1. Lactate binding is preferred with the more sterically demandingmono-anionic complexes and for [Eu.L⁹]⁻, the lactate/citrate ratio was30:1. These complexes were selected for further study (see sections Band C below), analysing the citrate or lactate content of serum, urine,saliva, seminal and prostate fluid samples using the luminescencemethod, comparing data obtained to measurements made using enzyme kits(obtained from Megazyme Ltd., Ireland).

B. Lactate Analysis in Various Biofluids

For lactate (and citrate) analyses using the Eu emission method, acalibration curve is first determined in an appropriate backgroundmedium, allowing the working range of the measurement to be defined.Thus, in a simulated prostate fluid background containing 0.3 mM HSA,0.1 M NaCl, 4 mM KCl, 3 mM NaHCO₃, 4 mM CaCl₂, 2 mM ZnCl₂, 5 mM MgCl₂(pH 6.5, 0.1 M HEPES), FIG. 1, modulation of Eu emission (10 μMconcentration) is evident over the lactate concentration range 0 to 1mM. Typically, a 5 μL sample of fluid, (seminal or prostate) is dilutedby a factor of 10, filtered through a 10 kD cut-off filter, and analysedin a 50 μL optical cuvette (λ_(exc) 336 nm). The intensity ratio of the692/619 or 613/622 or 613/619 emission bands in [Eu.L⁹]⁻ is measured andthe lactate concentration deduced by reference to the calibration curve.Samples of lactate in urine (3.5 mM), seminal fluid (3.8 mM) prostatefluid (4 mM) and reconstituted human serum (1.9 mM) were found to bewithin 10% of the concentration deduced enzymatically. In saliva, eachmethod gave a zero reading for lactate (±0.2 mM).

Excitation of the Eu(III)complex, [Eu.L⁹]⁻ in solution at 336 (±20) nmleads to an emission spectrum from 580 to 700 nm. The analysis utilisesratiometric detection, by plotting changes in the ratio of up to 4different wavelengths as a function of lactic acid concentration.Therefore, establishment of a suitable calibration curve and appropriatedilution provides a fast method for the determination of lactate invarious fluids or solutions. FIG. 3 shows high resolution Eu emissionspectra, of the europium complex [Eu.L⁹]⁻ (pH 6.5, 0.1 M HEPES, 298K,λ_(ex)=336 nm, C_(EuL)=10 μM) as a function of sodium lactateconcentration (using ISA Jobin-Yvon Spex Fluorolog-3 luminescencespectrometer), displaying (insert) the calibration curve determinedusing the stated emission band intensity ratios (613 vs. 618.5 nm) as afunction of added sodium lactate.

The precision of the measurement, which is an inherent feature of such aratiometric assay, generates less than 3% variance in the measuredintensity ratio for a given lactate concentration. Analyses were carriedout with a typical sample volume of 50 μL requiring about 1 μg of the Eucomplex. The sample preparation time and spectral acquisition is quick(<5 min.). A sensitive spectrofluorimeter is required possessing ±1 nmresolution. Inexpensive disposable cuvettes may be used and commerciallyavailable buffer solutions were used for dilution (pH 6.5). Analyseshave been undertaken for the determination of lactate in a variety ofbiological samples (plasma, seminal and prostate fluid, urine andsaliva), with confirmation of the accuracy of the analysis by comparisonwith the currently available (Megazyme Ltd., Ireland) enzymatic L-lacticacid assay kit (Table 2).

TABLE 2 Simultaneous analysis of unknown clinical samples (PR, SF, P, Sand U referring to Prostate Fluid, Seminal Fluid, Plasma, Saliva andUrine samples respectively); samples were measured in ×10 dilution.Sample [Eu.L⁹]⁻ Enzyme Kit PF-13007 4.0 ± 0.5 mM 3.6 ± 0.5 mM SF-R1 4.0± 0.5 mM 4.4 ± 0.4 mM P-A 1.8 ± 0.2 mM 1.9 ± 0.2 mM S-R1 0 mM 0 mM U-R11.0 ± 0.5 mM 1.2 ± 0.2 mMC. Determination of Citrate in Biological Fluids Using [Eu.L³]⁺

Similar procedures as described above in connection with thedetermination of lactate used to analyse for citrate using [Eu.L³]⁺. Inthis case, typically a 1 μL sample of prostatic fluid is diluted ×100.To obviate any interference due to changes in lactate concentration inprostate fluid samples, calibration measurements in this case only weremade (λ_(exc) 365 nm, 10 μs gate time, 50 μL optical cell) in thepresence of 0.1 M sodium lactate containing simulated prostate fluidsolution. Citrate was determined in fourteen samples of prostate fluid,and values ranging from 12 mM to 160 mM measured, confirmed (±10%) byindependent analysis (see FIG. 5) using the citrate lyase enzyme kit,for which the amount of bio-fluid required was 25 times greater. Usingsimilar methods, but with a 10-fold dilution of sample, citrate could bedetermined in the urine of healthy volunteers (range 3.5 to 5.5 mM(±10%)).

The europium complex, [Eu.L³]⁺ is based on a lanthanide-macrocycliccomplex, incorporating an azathiaxanthone antenna group, allowingexcitation of the Eu(III)complex in solution at 380 (±25) nm, leading tometal based emission from 580 to 710 nm. The analysis utilisesratiometric detection, plotting changes in the ratio of up to 6different emission wavelengths as a function of citric acidconcentration. Therefore, following establishment of a suitablecalibration curve and appropriate sample dilution a fast method isdefined for the determination of citrate in various fluids. FIG. 4 showshigh resolution Eu emission spectra, of the europium complex [Eu.L³]⁺(pH 6.5, 0.1 M HEPES, 298K, λ_(ex)=380 nm, C_(EuL)=10 μM) as a functionof sodium citrate concentration, displaying (insert left) thecalibration curve determined using the given emission band intensityratios (614 vs. 683 nm) as a function of sodium citrate. (insert right)time gated (10 μs) Eu emission spectra, of the europium complex (pH 6.5,0.1 M HEPES, 298K, λ_(ex)=365 nm, C_(EuL)=10 μM) as a function of sodiumcitrate concentration. FIG. 2 is similar (non inset) but is obtainedusing a 50 μs time-gate, 20 μm of [EuL³]⁺, λ_(exc)=365 nm, 0.1 M NaCl,0.3 mM NaHCO₃, 0.4 mM KCl, 0.03 mM HSA, 0.4 mM CaCl₂, 0.2 mM ZnCl₂, 0.5mM MgCl₂ and 0.1 M Na lactate.

The precision of the measurement, which is an inherent feature of such aratiometric assay, allows less than 3% variance in the measuredintensity ratio for a given citrate concentration. One analysis in atypical sample volume of 50 μL requires 1 μg of compound with rapidsample preparation and a fast (3 min) acquisition time. The samplerequirement of the analysis can be as small as 1 μL original fluid, as a100 times dilution is used with this method. Instrumental requirementsare a sensitive spectrofluorimeter possessing ±1 nm resolution (e.g. ISAJobin-Yvon Spex Fluorolog-3 or Ocean Optics Red Tide Spectrometer), anda commercially available buffer solution for dilution (pH 6.5). As theazathiaxanthone chromophore displays a significant amount of ligandfluorescence, causing interference in the form of a sloping baseline,time gated measurements are preferred for precise calibration. This canbe achieved using a time-gated spectrometer. In this case a home-builtinstrument was used, incorporating a time gating device. Using thisinstrument, the short-lived fluorescence of the sensitising moiety iseliminated by applying a 10 microsecond time-gate.

Using the calibration curve, citrate concentrations of clinical prostatefluid samples have been successfully differentiated using an appropriatecalibration curve.

D. Analyses of Citrate and Lactate in a Variety of Biological Samples(Plasma, Seminal and Prostate Fluid, Urine and Saliva) Using [Eu.L³]⁺and [Eu.L⁹]⁻

In order to demonstrate the application of [Eu.L³]⁺ in the analysis ofvarious bio-fluids for citrate, and the application of [Eu.L⁹]⁻ in theanalysis of various bio-fluids for lactate, analysis of clinical sampleswith unknown citrate and lactate levels has been undertaken.

To allow a comparison the citrate level in these samples was evaluatedusing a commercially available (Megazyme Ltd, Ireland) enzymatic citricacid kit. Typical analysis time using [Eu.L³] was 3 minutes with a 50 μLtotal sample volume (0.5 μL actual bio-fluid content) compared to the 20min acquisition time and 3 mL sample volume required using the enzymekit. Values refer to the mean of 3 measurements (s.d. in parenthesis,Table 2 below). Values are plotted in FIG. 5.

Analyses have also been undertaken for the determination of lactate in avariety of biological samples (plasma, seminal and prostate fluid, urineand saliva), with confirmation of the accuracy of the analysis bycomparison with the currently available (Megazyme Ltd., Ireland)enzymatic L-lactic acid assay kit (Table 2). Values are plotted in FIG.6.

TABLE 2 Citrate Lactate assay assay using the using the L-Lactate Samplepresent present dehidrogenase Citrate No. invention invention kit lyasekit 13007  11 ± 2 mM 4.0 ± 0.5 mM  3.6 ± 0.5 mM  15 ± 3 mM 103007 110 ±1O mM 2.5 ± 0.5 mM — 102 ± 5 mM 103009 157 ± 1O mM 4.0 ± 0.5 mM — 152 ±5 mM I-N  15 ± 3 mM 3.1 ± 0.5 mM — — 3-N 110 ± 1O mM 6.0 ± 0.5 mM — — 1 20 ± 3 mM 6.4 ± 0.2 mM  6.1 ± 0.2 mM  23 ± 3 mM 2  16 ± 2 mM 6.5 ± 0.2mM  6.3 ± 0.2 mM  11 ± 3 mM 3  12 ± 2 mM 6.1 ± 0.2 mM  6.1 ± 0.2 mM  14± 3 mM 4  86 ± 5 mM — —  90 ± 5 mM 5 148 ± 5 mM — — 146 ± 5 mM 6  99 ± 5mM — — 102 ± 5 mM 7  46 ± 5 mM 6.2 ± 0.2 mM 5.7* ± 0.2 mM  40 ± 5 mM 8152 ± 5 mM — — 151 ± 5 mM 9  88 ± 5 mM — —  94 ± 5 mM 10  22 ± 3 mM 2.7± 0.2 mM —  18 ± 3 mM 13 112 ± 5 mM — — 105 ± 5 mM 15  22 ± 3 mM 5.4 ±0.2 mM —  19 ± 3 mM SeminalF  47 ± 5 mM 3.8 ± 0.3 mM 4.4# ± 0.5 mM  42 ±5 mM Urine  5 ± 2 mM 3.5 ± 0.3 mM  3.3 ± 0.3 mM  5 ± 1 mM Plasma   — 1.9± 0.2 mM  1.9 ± 0.2 mM — Saliva   — O mM O mM — Samples 13007, 103007,103009, 1-N, 3-n and 1-15 all of prostatic fluid; Seminal F = seminalfluid; for citrate measurements: ×200 dilution; for lactate measurements×10 dilution.

The invention claimed is:
 1. A compound of the following formula:

wherein each -A is —CO₂H, or a salt thereof; and R³ is either hydrogen or methyl.
 2. The compound of claim 1, wherein R³ is hydrogen.
 3. The compound of claim 1, wherein R³ is methyl.
 4. A complex comprising: a lanthanide (III) ion; and a compound of the following formula

wherein each -A is —CO₂H, or a salt thereof; and R³ is either hydrogen or methyl.
 5. The complex of claim 4, wherein R³ is hydrogen.
 6. The complex of claim 4, wherein R³ is methyl.
 7. The complex of claim 4 wherein the lanthanide (III) ion is europium (III) or terbium (III).
 8. The complex of claim 4 wherein the lanthanide (III) ion is europium (III).
 9. A method of analyzing lactate present in a sample of interest, the method comprising: (i) contacting the sample of interest with a complex as defined in claim 4; (ii) exciting the azaxanthone; and (iii) determining the quantity or concentration of any lactate in the sample of interest by analysis of the modulation in one or more emission bands resultant from the exciting where citrate is present.
 10. A method of analyzing lactate present in a sample of interest, the method comprising: (i) contacting the sample of interest with a complex as defined in claim 7; (ii) exciting the azaxanthone; and (iii) determining the quantity or concentration of any lactate in the sample of interest by analysis of the modulation in one or more emission bands resultant from the exciting where citrate is present.
 11. A method of analyzing lactate present in a sample of interest, the method comprising: (i) contacting the sample of interest with a complex as defined in claim 8; (ii) exciting the azaxanthone; and (iii) determining the quantity or concentration of any lactate in the sample of interest by analysis of the modulation in one or more emission bands resultant from the exciting where citrate is present.
 12. A method of, or for use in, the diagnosis of excessive lactic acid comprising: (i) obtaining a liquid sample from a subject; (ii) optionally diluting the liquid sample; and (iii) practicing a method as defined in claim 9, wherein the liquid sample or the diluted liquid sample constitutes the sample of interest.
 13. The method of claim 12 wherein the liquid has been previously obtained from the subject. 